Axon regeneration remains a major roadblock in functional recovery after nervous system injury. Recent studies suggest that regeneration capacity of injured axons is neuronal subtype-specific, but its molecular and cellular determinants remain unknown. Nerve damage triggers complex injury responses in neurons and the surrounding glial cells and macrophages, but how these diverse cell types signal to each other to coordinately regulate axon regeneration is largely unknown. Our long-term goal is to uncover the repertoire of pro- and anti-regeneration factors for designing combinatorial therapy to promote axon regeneration. To fulfill this goal, we have established a sensory neuron axotomy model in Drosophila, which resembles mammalian regeneration models at the phenotypical and molecular levels. In particular, Drosophila sensory neurons display neuronal subtype-specific regeneration. Our model allows the molecular, morphological and physiological characterization of injured neurons, and the surrounding glia and macrophages, at the single-cell resolution. Recent studies have suggested that increase of neuronal activity promotes axon regeneration after injury, but it is unclear whether and how nerve injury alters the activity of neurons, glial cells and macrophages, and how the collective activities in these diverse cell types contribute to axon regeneration. Our objective in current proposal is to determine how glia and macrophages interact with neurons to regulate neuronal subtype-specific firing and regeneration. Specifically, we will test the hypothesis that 1) axotomy-induced neuronal burst firing mediates neuronal subtype-specific regeneration; 2) glia and macrophages signal to injured neurons to regulate neuronal firing and regeneration. In this proposal, we plan to fully leverage the sophisticated Drosophila genetics with state-of-art techniques, including highly reproducible single-neuron axotomy, optogenetics, in vivo time-lapse GCaMP imaging to simultaneously monitor Ca2+ activities in neurons and glia/macrophages, single-neuron transcriptional analysis, to address our hypothesis.